Recently we have demonstrated an essential role of the lens epithelium-derived growth factor (LEDGF/p75) in the human immunodeficiency virus type 1 (HIV-1) DNA integration process. T cells lacking this chromatin-bound protein are resistant to HIV-1 infection at the viral integration step suggesting LEDGF/p75 as a target for anti-HIV drug development. Although we demonstrated that LEDGF/p75 chromatin and integrase binding domains are required, its exact role in this process is still unknown. The goal of this proposal is to understand the molecular mechanism of LEDGF/p75 HIV-1 DNA integration. This knowledge will have a direct impact in the development of drugs targeting this process. Our results will help the design of high-throughput screening methods for small-molecule HIV integration inhibitory compounds. Specific Aims (1) To determine if LEDGF/p75 is required for chromatin tethering of HIV pre-integration complexes (PICs). LEDGF/p75 determines chromatin localization of integrase expressed as a sole protein by plasmid transfection in non-infected cells. It has been proposed that LEDGF/p75 acts as a molecular tether between integrase and chromatin. A tethering role of LEDGF/p75 in HIV integration was also postulated because of the requirement of the same LEDGF/p75 domains for integrase localization and HIV integration. However, this model awaits direct evidence for demonstration. Using chimeric LEDGF proteins, subcellular fractionation and DNA damage signaling, we will evaluate this model in HIV infected cells. (2) To evaluate the interaction of LEDGF/p75 with DNA repair proteins. The interaction of LEDGF/p75 with DNA repair proteins will be investigated by GST pull-down experiments. Relevant domains in LEDGF/p75 will be mutated and its relevance in HIV infection evaluated. (3) Development of a high-throughput screening system for small inhibitory compounds of LEDGF/p75-integrase interaction. LEDGF/p75 protects integrase from proteasomal-mediated degradation. Integrase-eGFP fusion protein will be expressed in HeLa cells;drugs interfering with LEDGF/p75-integrase interaction are expected to reduce eGFP expression in these cells.